Gel electrophoresis, a commonly used method on molecular biology research, is a technique designed to separate, identify and purify DNA, RNA and protein molecules based on their weight, size and shape. This technique, which is simple and rapid to perform, is carried out by first preparing a gel. When the gel is ready it is placed in a gel box, immersed in a buffer solution, and connected to a power source. Once stimulated by the electric field that is set up in the gel, the molecules move through the gel matrix at different rates. The migration rate for each species of molecule is dependent upon the electrical charge, the size and shape of the molecules, as well as on the composition of the gel. Most commonly, the smaller molecules will move through the matrix at a quicker pace than those of a larger size. Sufficient quantity of buffer (typically TAE, TBE or protein running buffer) is generally used to ensure that the electric field is set up in the gel, and that the gel is covered with it and thus prevent the gel from drying out during electrophoresis. When loading a sample containing the molecule species of interest into the gel, a loading dye is typically used. The loading dye normally allows easy visualization of the solution during the loading process, as well as enabling the density of the sample to be increased to ensure that the sample is fully and evenly accommodated in a corresponding well in the gel, and further allows visualisation of the migration during electrophoresis.
The most commonly used gels are prepared with either agarose or acrylamide, either one of which can be provided in varying shapes, sizes and thicknesses. The deciding factor as to which particular gel and its physical attributes is generally related to the size of molecule being separated.
Acrylamide is usually chosen for relatively small molecules such as proteins, while agarose is used for larger molecules such as DNA or RNA. In any case, while agarose is the preferred choice for horizontal gel electrophoresis, being cast in typically open trays, acrylamide is used typically used only for vertical gel electrophoresis, being cast between two glass plates, and is not used for horizontal electrophoresis in the art. The reason for this is that acrylamide does not tend to provide a mechanically stable well structure because the acrylamide gel is very elastic and its thickness is very thin (up to 1.5 mm), and if used for horizontal gel electrophoresis adjacent wells tend to cave in negating any possibility to load samples therein. When acrylamide gels are used in vertical electrophoresis, the depth dimension of the wells is aligned with the electric field, and relatively deep and narrow wells may be provided which are substantially well-spaced with respect to neighbouring wells. Furthermore, in vertical electrophoresis the wells are created between two parallel spaced vertical glass plates, which by providing two opposed walls for each well enable the wells to remain stable. However, vertical gel electrophoresis is not without problems. For example, it is easier to load samples into horizontal gel wells than in vertical gel wells. It is also difficult to cast vertical acrylamide gels, particularly gradient gels, and special loading tips for the samples are required. In vertical gel electrophoresis, one buffer tank is vertically spaced from the other buffer tank, and it often occurs that running buffer will leak from the upper tank to the lower tank; eventually, the upper tank gets drained of buffer severing electrical connection between the upper electrodes and the gel.
Unpolymerised acrylamide is a potent neurotoxin and is absorbed through the skin. The effects of acrylamide are cumulative. Although polymerised acrylamide is considered to be non-toxic, it should nevertheless be handled with care because of the possibility that it may contain small quantities of unpolymerised acrylamide. Thus, contact with users must be strictly avoided, particularly when the acrylamide is unpolymerised and/or is in powder form, during handling and when disposing of the gels after use.
It is therefore an aim of the present invention to provide a device and method which overcomes the limitations of prior art electrophoresis devices and methods.
It is another aim of the present invention to provide an acrylamide-based gel that may be used for horizontal electrophoresis.
It is another aim of the present invention to provide a device for enabling safe handling and disposal of such gels which may contain harmful substances.
It is another aim of the present invention to provide such a device that is simple to use.
It is another aim of the present invention to provide such a device that is relatively simple mechanically and thus economic to produce.
These and other aims are accomplished in the present invention by providing a precast-gel cassette for horizontal electrophoresis, in particular a disposable and closed cassette for horizontal electrophoresis. In particular, the cassette enables the use of acrylamide gels in horizontal electrophoresis. This is accomplished by precasting a transverse strip of agarose in ionic communication with the main body of acrylamide gel within the cassette. The agarose strip enables stable wells to be formed therein (by means of a comb, for example) for insertion of samples therein. Such a agarose/acrylamide hybrid gel combination is often difficult to cast in situ by users, and the acrylamide portion thereof poses safety issues during handling and disposal of the gel. Accordingly, the present invention further provides significant advantages to users wishing to use acrylamide gels for horizontal electrophoresis, since the cassette comes prepared with precast acrylamide gel and agarose gel included therein.
In another aspect of the invention, the cassette comprises at least one and preferably a pair of agarose plugs one at each longitudinal open end of the cassette. The plugs essentially isolate the acrylamide gels from the outside of the cassette, thereby minimising any possibility of human contact with the acrylamide gel in the cassette. This is an important safety feature, particularly in view of the disposability of the cassette, which thus minimises any handling of toxic substances.
In the preferred embodiment, the cassette comprises a box-like construction, having a bottom flat base and four vertical walls joined thereto about its periphery, and an upper cover mountable onto the vertical walls to define a gel chamber into which gel may be precast. The cassette also comprises openings at two opposite ends of the bottom base to enable ionic communication between the gel and an electrolytic solution in which the cassette may be partially immerged. The openings are preferably comprised in hollow legs running the transverse length of the cassette at two longitudinal ends thereof, the legs comprising gel in ionic communication with the main body of gel within the cassette. This design is particularly adapted for using the cassette with standard ion exchange chambers. An agarose/acrylamide hybrid gel is provided within the chamber for performing horizontal electrophoresis, and agarose gel is provided in the legs to provide ionic communication between the hybrid gel and the external buffer solutions, while at the same time providing a safety barrier between the acrylamide gel within the cassette and the outside thereof.
U.S. Pat. No. 3,888,759 discloses a gel cassette having a substantially box-like construction, having a downwardly depending transversely extending hollow leg at each longitudinal end of the cassette. The device appears to be reusable, providing the user with different options, and it appears intended for the user to cast the gel each time, rather than providing a precast package. There is, however, no disclosure or any indication of providing a hybrid gel for enabling horizontal electrophoresis with an acrylamide gel.
U.S. Pat. No. 5,443,704 discloses a substantially box-like container assembly for an electrophoresis gel, containing more than one precast gel deposited therein. However, the different gels are provided in a stacked arrangement, which does not provide the advantages of the present invention at least in terms of enabling an acrylamide gel to be used for horizontal electrophoresis in combination with a juxtaposed agarose strip comprising then wells.
U.S. Pat. No. 5,064,769 discloses a gel for immunoassay of a single protein species in which the horizontal gel comprises a first part made from acrylamide gel having a proportion of agarose (0.7%) sufficient to enable stable wells to be formed therein. The first part of the gel is juxtaposed with a second part made from agarose gel. Acrylamide gel is used in this reference only to eliminate diffusion effects in the wells, and is not used, nor can it be used successfully, to form the wells—this is accomplished by the 0.7% agarose. Use of the agarose gel in the second part of the gel combination is well known for horizontal electrophoresis. The gel arrangement provided by this reference is in fact the opposite way round to the present invention, and thus teaches away from the present invention. There is no disclosure or suggestion regarding using acrylamide for horizontal gel electrophoresis, or of using agarose therewith for providing the wells.
In U.S. Pat. No. 3,930,983 an arrangement and process are disclosed for determining antigens, in which a support plate is coated with an agar or agarose as a matrix in successive gel strips. However, there is no disclosure or suggestion that other than the first strip containing the wells the other strips should be made from acrylamide gel rather than agarose. In fact, it appears that the gel should be the same for all the strips, the only variable being the monospecific antiserum contained in each of the strips.
U.S. Pat. No. 5,582,702 is directed to a self-contained electrophoresis apparatus comprising a housing having a gel body accommodated therein together with ion exchange matrices and electrodes, which are electrically connectable to an external power source. The apparatus is thus not generally compatible with existing ion exchange chambers currently used for horizontal electrophoresis. While it is stated therein that the gel may be from agarose or acrylamide, there is no disclosure or suggestion of how to overcome the well integrity problem encountered with acrylamide gels when it is attempted to use the same for horizontal electrophoresis. In particular, this problem is not addressed by the reference, and there is no disclosure or suggestion of how to overcome the same, less so in the manner of the present invention.
WO 95/20155 relates to a sample holder in the form of a well, into which a sample and a first molten gel is introduced. When the first gel/sample mixture has solidified, the sample holder is applied against one end of a second gel slab, such as to bring the first gel/sample solidified mixture in ionic contact with the second gel. At no time is the first gel in solidified form brought into contact with the second gel prior to introducing the sample. Thus, the first gel is in no way adapted for accommodating the sample therein when in the solidified state, and thus neither discloses nor anticipates the present invention. The method and apparatus disclosed by the patent still requires the first gel to be cast (mixed with sample), and further manipulation thereof, in contrast to the present invention in which the first gel and the second gel may be precast, and are ready (when solidified) to accommodate therein samples, typically via wells in the first gel.
WO 99/30145 relates to a slotted electrophoresis gel composition and methods for use, for providing a multilayered gel for vertical gel electrophoresis. It does not address, nor provide a solution for, the problem of forming stable sample wells for horizontal electrophoresis in an acrylamide gel. Specifically, it does not disclose nor suggest a hybrid gel as in the present invention, but merely a slotted gel structure having at least three layers:—two primary gel layers on either side of a slotted second gel layer.
EP 471949 discloses a capillary tube for performing capillary zone electrophoresis. The tube is modified by including a polystyrene frit that divides the tube into a downstream free zone, and an upstream zone which can comprise a polyacrylamide stacking gel. The gel plug functions as a filter to pre-treat the samples that are to be analysed in the free zone of the tube.
WO92/17259 describes a method for identifying a solute of interest in an effluent stream. A sample containing the mixture to be separated is passed through a first system capable of partitioning the components of the mixture, and a detector provides a first output that describes the temporal and/or spatial sequence of components exiting the first system. The effluent stream is then passed through a second system capable of extracting a solute of interest from the effluent, and a detector provides a second output that describes the temporal and/or spatial sequence of components exiting the second system, which no longer includes the solute of interest. The solute of interest can then be identified in the first output by comparing this to the second output. This method is thus directed at identifying a substance in a first separating system by employing a parallel second separating system.
Other publications of background interest include EP 971229, U.S. Pat. No. 5,228,971, WO 95/14921, DE 3232685, EP 199470, U.S. Pat. Nos. 5,827,418, 3,803,020, WO 98/10277 and U.S. Pat. No. 3,873,433.